The Structural Diversity of Marine Microbial Secondary Metabolites Based on Co-Culture Strategy: 2009–2019

Marine microorganisms have drawn great attention as novel bioactive natural product sources, particularly in the drug discovery area. Using different strategies, marine microbes have the ability to produce a wide variety of molecules. One of these strategies is the co-culturing of marine microbes; if two or more microorganisms are aseptically cultured together in a solid or liquid medium in a certain environment, their competition or synergetic relationship can activate the silent biosynthetic genes to produce cryptic natural products which do not exist in monocultures of the partner microbes. In recent years, the co-cultivation strategy of marine microbes has made more novel natural products with various biological activities. This review focuses on the significant and excellent examples covering sources, types, structures and bioactivities of secondary metabolites based on co-cultures of marine-derived microorganisms from 2009 to 2019. A detailed discussion on future prospects and current challenges in the field of co-culture is also provided on behalf of the authors’ own views of development tendencies.


Introduction
Although many industrial sectors have stopped their dependence on natural product (NP) drug discovery programs, NPs are still of great interest to many pharmaceutical communities and are important sources of bioactive compounds [1,2]. Marine microbes, as an important source of bioactive NPs, have elicited widespread attention [3][4][5]. However, the discovery of novel marine microbial NPs is becoming more difficult and the rate of rediscovery of known NPs is being gradually increased. On the other hand, recent genomic sequencing has revealed the presence of numerous biosynthetic gene clusters in some microbes that may be responsible for the biosynthesis of NPs which are not found under classical cultivation conditions [6,7]. Therefore, many alternative strategies have been explored to activate these silent and cryptic biosynthetic genes. The co-culturing of marine microbes involves the culturing of two or more marine microbes together on/in certain conditions; microorganisms can communicate with each other through direct or indirect contact, thereby stimulating the silent gene clusters to produce special NPs [2,8] (Figure 1). This strategy can promote the production of complex and novel skeletons with numerous stereocenters [9][10][11]. Hence, the co-culturing of marine microbes draws widespread attention in the scientific community as a potential source of unknown

Alkaloids
The nitrogenous alkaloids represented the most abundant class of compounds that were produced by the co-cultures of marine microorganisms with diverse skeletons and biological activities [15,16]. Eighty alkaloidal metabolites were isolated and identified from different microbial environments ( Figure 2B), and the co-cultures of marine fungi-bacteria represented 51% of the total isolates ( Figure  3).

Alkaloids
The nitrogenous alkaloids represented the most abundant class of compounds that were produced by the co-cultures of marine microorganisms with diverse skeletons and biological activities [15,16]. Eighty alkaloidal metabolites were isolated and identified from different microbial environments ( Figure 2B), and the co-cultures of marine fungi-bacteria represented 51% of the total isolates ( Figure 3).
Compounds 2, 6, 8, 13 and 14 inhibited the proliferation of the human prostate cancer cells 22Rv1 at 100 µM. Notably, 2 and 13 drastically reduced the viability of 22Rv1 prostate cancer cells at 10 µM by 25% and 55%, respectively. 22Rv1 cancer cell lines were resistant to hormone therapy at conventional chemotherapy including two new 2nd generation drugs enzalutamide and abiraterone owing to the presence of the androgen receptor splice variant-7 (AR-V7). Therefore, the active NPs drugs in these cells might be further investigated in the treatment of different human drug-resistant prostate cancer. 6 and 7 displayed weak cytotoxicity against HeLa and L1210 cell lines with half maximal inhibitory concentration (IC 50 ) in the range of 22-52 µg/mL [18]. Although 6 and 7 had the similar structure with 9, compound 9 did not display the similar cytotoxic activity against HeLa and L1210 cell lines. The significant difference in cytotoxicity might be attributed to the possible existence of pyrroloindole system in 9 rather than the dihydroxypyrano-2-oxindole ring system of 6 and 7 [18,19]. In addition, compounds 1, 2, 5, 9, 13 and 14 did not exhibit any cytotoxicity against human non-malignant (HEK 293 T and MRC-9) or malignant (PC-3, LNCaP, and 22Rv1) cell lines at concentrations up to 100 µM for 48 h [17].
The co-fermentation of marine mangrove epiphytic fungi Aspergillus sp. FSY-01 and FSW-02 collected from a rotten fruit of mangrove Avicennia marina in Zhanjiang, Guangdong Province, China, yielded a new alkaloid, aspergicin (15), together with two known secondary metabolites, neoaspergillic acid (16) and aspergicine (17) (Figure 5) [20,21]. Notably, compounds 17 and 15 are chemically isomers, and consequently aspergicine (17) may be the precursor of aspergicin (15) through a proton 1, 2-shift [22]. Compounds 2, 6, 8, 13 and 14 inhibited the proliferation of the human prostate cancer cells 22Rv1 at 100 μM. Notably, 2 and 13 drastically reduced the viability of 22Rv1 prostate cancer cells at 10 μM by 25% and 55%, respectively. 22Rv1 cancer cell lines were resistant to hormone therapy at conventional chemotherapy including two new 2nd generation drugs enzalutamide and abiraterone owing to the presence of the androgen receptor splice variant-7 (AR-V7). Therefore, the active NPs drugs in these cells might be further investigated in the treatment of different human drug-resistant prostate cancer. 6 and 7 displayed weak cytotoxicity against HeLa and L1210 cell lines with half maximal inhibitory concentration (IC50) in the range of 22-52 μg/mL [18]. Although 6 and 7 had the similar structure with 9, compound 9 did not display the similar cytotoxic activity against HeLa and L1210 cell lines. The significant difference in cytotoxicity might be attributed to the possible existence of pyrroloindole system in 9 rather than the dihydroxypyrano-2-oxindole ring system of 6 and 7 [18,19]. In addition, compounds 1, 2, 5, 9, 13 and 14 did not exhibit any cytotoxicity against human non-malignant (HEK 293 T and MRC-9) or malignant (PC-3, LNCaP, and 22Rv1) cell lines at concentrations up to 100 μM for 48 h [17].
Cytochalasans were fungal metabolites that were structurally identified by the presence of a reduced isoindone nucleus connected with a macrocyclic ring [44]. Six cytochalasans (50)(51)(52)(53)(54)(55) showed strong toxicity against Streptomyces sp. with 50-80% inhibition at 2-16 µg/mL, and most of them even exhibited 60% inhibition at 2 µg/mL, but they had no any effect on the fungus A. flavipes at the same concentration. This indicated that cytochalasans could help A. flavipes to compete with Streptomyces sp., which was an important support for their potential ecological role. All cytochalasans also exhibited obvious toxicity against human cell lines, as cytochalasans had the ability to inhibit, specifically, the actin filament elongation by blocking the polymerization sites [45][46][47]. Thus, all six compounds (50-55) exhibited powerful toxicity against Streptomyces sp. at 2-16 µg/mL with inhibition rate of 50-80%. Notably, most of these compounds displayed strong inhibitory activity with inhibition rate of 60% even at 2 µg/mL, whereas none of them had antimicrobial activity against the marine-derived producer A. flavipes at the same concentration. These findings implied that the co-culture through microbial physical contact could stimulate the expression of silent gene cluster that was responsible for the production of cytochalasans.
The cyclic siderophore, nocardamine (58), had inhibitory effects on the proliferation of human tumor cell lines: SK-Mel-5 with an IC 50 value of 18 µM, T-47D with an IC 50 value of 6 µM, PRMI-7951 with an IC 50 value of 14 µM and SK-Mel-28 with an IC 50 value of 12 µM [48]. Compared with the pure cultures, some novel metabolites were observed in the mixed culture. Two fungal prenylated indole metabolites, 56 and 59, which were not traced before in A. fumigatus, were induced. Both of them had an oxazino [6,5-b]indole nucleus which was not previously found in nature. Additionally, the yield of compound 61 was obviously higher than that of the monoculture of Streptomyces leeuwenhoekii C58. It was the first time that a bi-lateral cross talk was proved, which resulted in dual induction of both fungal and bacterial metabolites in the same culture conditions. 64 displayed cytotoxic activities toward mouse lymphoma cell line L5178Y with an IC 50 value of 16.7 µM and in vitro toward testosterone-dependent prostate LNCaP cells with an IC 50 value of 2.1 µM [49].

Alkaloids Derived from the Co-Cultures of Different Marine Bacteria
Thirteen alkaloids were isolated from the co-culture of different marine bacteria ( Figures 2E and 3); the structures of these isolates were listed in Figure 10. The average yields of five known tryptamine derivatives, N-acetyltryptamine (68), N-propanoyltryptamine (69), bacillamide C (70), bacillamide B (71) and bacillamide A (72) using the co-fermentation of marine strain Streptomyces sp. CGMCC4.7185 and Bacillus mycoides isolated from marine sediments of the Nanji Island (China, 27 • 42 N, 121 • 08 E), were 14.9, 2.8, 9.6, 13.7 and 3.0 mg/L, respectively, which were all undetectable under simple culture conditions [50]. This was the first report of applying a microorganism co-culture system to enhance the yields of known compounds [50].
Pim-1 kinase is a well-established oncoprotein in several tumor entities, such as prostate cancer, pancreatic cancer, colorectal cancer and myeloid leukemia. Inhibition of Pim-1 kinase would prevent the growth of tumor cells. Compounds 73 and 75 exhibited potent Pim-1 kinase inhibitors with IC 50 values of 0.3 µM and 0.946 µM, respectively. Docking studies showed the binding model of 73 and 75 in the ATP pocket of Pim-1 kinase. They also exhibited obvious antiproliferative activity against human promyelocytic leukemia HL-60 (IC 50 2.8 and 4.9 µM) and human colon adenocarcinoma HT-29 (IC 50 3.6 and 3.7 µM). This indicated that 73 and 75 could act as potential Pim-1 kinase inhibitors that mediated the inhibitory effects on the growth of tumor cells [51].
In addition, only compound 79 was documented against Trypanosoma brucei (IC50 19 μΜ), Bacillus sp. (11 mm inhibition zone diameter) and Actinokineospora sp. EG49 (15 mm inhibition zone diameter) [52]. The yield of 79 was very high in the co-culture process. However, it was not detected in the single microbial culture. Co-culture strategy not only enhanced the chemical diversity of the metabolites but also increased the production of metabolites undetected in the single microbial culture.

Anthraquinones
Thirteen different anthraquinone isolates were obtained from different marine microbial cocultures; the co-cultures of marine fungi-bacteria represented the majority, 69% (9/13 isolates; Figures  2B and 11). In addition, only compound 79 was documented against Trypanosoma brucei (IC 50 19 µM), Bacillus sp. (11 mm inhibition zone diameter) and Actinokineospora sp. EG49 (15 mm inhibition zone diameter) [52]. The yield of 79 was very high in the co-culture process. However, it was not detected in the single microbial culture. Co-culture strategy not only enhanced the chemical diversity of the metabolites but also increased the production of metabolites undetected in the single microbial culture.

Anthraquinones
Thirteen different anthraquinone isolates were obtained from different marine microbial co-cultures; the co-cultures of marine fungi-bacteria represented the majority, 69% (9/13 isolates; Figures 2B and 11). In the recent study, the combination of cultures from two different developmental stages of marine alga-derived Aspergillus alliaceus (teleomorph: Petromyces alliaceus) drastically changed the metabolite profile and resulted in the production of allianthrone A (81) and two diastereomers,

Anthraquinones Derived from the Co-Cultures of Different Marine Fungi
In the recent study, the combination of cultures from two different developmental stages of marine alga-derived Aspergillus alliaceus (teleomorph: Petromyces alliaceus) drastically changed the metabolite profile and resulted in the production of allianthrone A (81) and two diastereomers, allianthrones B (82) and C (83) (Figure 12) [53]. 81-83 exhibited cytotoxic activity against SK-Mel-5 melanoma cell lines with IC 50 (11.0, 12.2, and 19.7 µM) and HCT-116 colon carcinoma cells with IC 50 (9.0, 10.5 and 13.7 µM), respectively. This study presented the first example of elicitation of novel fungal chemical diversity by a co-existing strategy of two different developmental phenotypes of Aspergillus species. For several Aspergilli, e.g., A. alliaceus, asexual and sexual life developmental stages were known. However, rarely did they co-cultivate at the same time. Even more surprising was the presence of novel bianthrones when the sclerotial and asexual morphs of the same species co-existed. There were only a few examples that showed differences in secondary metabolites in fungi based on their distinct developmental stages or chemical profiles for the two mating types of heterothallic fungi. However, none of these compounds displayed any activity against P. aeruginosa, E. faecium, S. aureus, E. coli, C. albicans and B. subtilis. Furthermore, non-significant results were obtained against lung (A549), prostate (PC3) and breast (MCF-7) human cancer cells compared with the positive control, etoposide [53]. Figure 11. Anthraquinones isolated from the co-cultures of marine fungi-fungi, fungi-bacteria and bacteria-bacteria.

Anthraquinones Derived from the Co-Cultures of Different Marine Fungi
In the recent study, the combination of cultures from two different developmental stages of marine alga-derived Aspergillus alliaceus (teleomorph: Petromyces alliaceus) drastically changed the metabolite profile and resulted in the production of allianthrone A (81) and two diastereomers, allianthrones B (82) and C (83) (Figure 12) [53]. 81-83 exhibited cytotoxic activity against SK-Mel-5 melanoma cell lines with IC50 (11.0, 12.2, and 19.7 μM) and HCT-116 colon carcinoma cells with IC50 (9.0, 10.5 and 13.7 μM), respectively. This study presented the first example of elicitation of novel fungal chemical diversity by a co-existing strategy of two different developmental phenotypes of Aspergillus species. For several Aspergilli, e.g., A. alliaceus, asexual and sexual life developmental stages were known. However, rarely did they co-cultivate at the same time. Even more surprising was the presence of novel bianthrones when the sclerotial and asexual morphs of the same species co-existed. There were only a few examples that showed differences in secondary metabolites in fungi based on their distinct developmental stages or chemical profiles for the two mating types of heterothallic fungi. However, none of these compounds displayed any activity against P. aeruginosa, E. faecium, S. aureus, E. coli, C. albicans and B. subtilis. Furthermore, non-significant results were obtained against lung (A549), prostate (PC3) and breast (MCF-7) human cancer cells compared with the positive control, etoposide [53].  subtilis [38]. Versiconol (87) was characterized as an inhibitor of protein tyrosine kinases against EGF-R and v-abl protein tyrosine kinases that were responsible for catalyzing phosphorylation of tyrosine residues of protein substrates, and suppression of MK-cells [54].  Versiconol (87) was characterized as an inhibitor of protein tyrosine kinases against EGF-R and v-abl protein tyrosine kinases that were responsible for catalyzing phosphorylation of tyrosine residues of protein substrates, and suppression of MK-cells [54]. 89 displayed inhibitory activity against the Gram-positive S. aureus with MIC value of 50 µM and antifungal activity against Fusarium solani with MIC values of 16-32 µg/mL [38,55]. The cytotoxic bioassay of 90 was recorded against mouse lymphoma cell line L5178Y with an IC 50 value of 21.2 µM. Moreover, 91 displayed antibacterial activity against B. subtilis (MIC = 8-16 µg/mL) and the Gram-positive S. aureus (MIC = 25 µM) and four Gram-positive microbes, including two E. faecalis and two E. faecium (MIC = 12.5-25 µM) [38,55]. Neither 89 nor 91 had cytotoxicity against L5178Y cell line, which implied that their antimicrobial activities were not associated with their respective general toxicities. Besides, 90 also displayed mild cytotoxic activity against human lung cancer cells H460 and the human prostate cancer cells PC-3 with IC 50 values of 27.2 and 19.5 µM, respectively [56]. Other compounds did not exhibit distinct cytotoxic activity against L5178Y cell line and antibacterial activity against five Gram-positive microbes, including one S. aureus, two E. faecalis and two E. faecium.

Anthraquinones Derived from the Co-Cultures of Different Marine Bacteria
A new antibiotic, keyicin (93) (Figure 14), was purified and identified from a co-culture of two marine invertebrate-associated bacteria Micromonospora sp. WMMB-235 and Rhodococcus sp. WMMA-185 [57]. It showed selective inhibitory activity against Gram-positive bacteria and could inhibit the growth of B. subtilis and Methicillin Sensitive Staphylococcus aureus (MSSA) with MIC values of 9.9 µM and 2.5 µM, respectively. In contrast to many anthracyclines, 93 might modulate fatty acid metabolism and exhibit antibacterial activity without nucleic acid damage that is explained by keyicin's mechanism of action (MOA) based on E. coli chemical genomics studies [57].

Cyclopeptides
Cyclopeptides are cyclic compounds mainly formed by the amide bonds of proteinogenic or non-proteinogenic amino acids bound together. Several fungal cyclic peptides have been developed as pharmaceuticals, such as the echinocandins, pneumocandins and cyclosporin A [58]. Six cyclopeptides were produced by the co-cultures of marine fungi-fungi (four isolates, 67%) and fungibacteria (two isolates, 33%) from different marine sources. However, marine bacteria-bacteria did not yield these structures in this period of investigation.

Cyclopeptides
Cyclopeptides are cyclic compounds mainly formed by the amide bonds of proteinogenic or non-proteinogenic amino acids bound together. Several fungal cyclic peptides have been developed as pharmaceuticals, such as the echinocandins, pneumocandins and cyclosporin A [58]. Six cyclopeptides were produced by the co-cultures of marine fungi-fungi (four isolates, 67%) and fungi-bacteria (two isolates, 33%) from different marine sources. However, marine bacteria-bacteria did not yield these structures in this period of investigation.  (Figure 15) [60] were identified from the co-culture of two mangrove fungi Phomopsis sp. K38 and Alternaria sp. E33 isolated from the South China Sea. Meanwhile, the co-cultivation of two marine alga-derived fungi Aspergillus sp. BM-05 and BM-05ML isolated from a brown algal species collected off Helgoland, North Sea, Germany, yielded a new cyclotripeptide, psychrophilin E (97) (Figure 15) [30]. bacteria (two isolates, 33%) from different marine sources. However, marine bacteria-bacteria did not yield these structures in this period of investigation.

Cyclopeptides Derived from the Co-Cultures of Marine Fungi and Bacteria
Recently, the chemical investigation of the mixed-fermentation of a marine fungus Aspergillus versicolor isolated from the sponge Agelas oroides and B. subtilis yielded two cyclic pentapeptides, one new cotteslosin C (98) and a known cotteslosin A (99) (Figure 16) [38]. Both of them did not show significant cytotoxic activity towards mouse lymphoma cell line L5178Y, or even antibacterial activity against five Gram-positive microbes, including one S. aureus, two E. faecalis and two E. faecium [38]. 99 displayed weak cytotoxicity against another three human cancer cell lines, prostate DU145, melanoma MM418c5 and breast T47D, with EC 50 values of 90, 66 and 94 µg/mL, respectively [61].

Cyclopeptides Derived from the Co-Cultures of Marine Fungi and Bacteria
Recently, the chemical investigation of the mixed-fermentation of a marine fungus Aspergillus versicolor isolated from the sponge Agelas oroides and B. subtilis yielded two cyclic pentapeptides, one new cotteslosin C (98) and a known cotteslosin A (99) (Figure 16) [38]. Both of them did not show significant cytotoxic activity towards mouse lymphoma cell line L5178Y, or even antibacterial activity against five Gram-positive microbes, including one S. aureus, two E. faecalis and two E. faecium [38]. 99 displayed weak cytotoxicity against another three human cancer cell lines, prostate DU145, melanoma MM418c5 and breast T47D, with EC50 values of 90, 66 and 94 μg/mL, respectively [61].

Macrolide
There were no reported macrolides from the co-cultures of marine fungi-fungi and fungibacteria. Only one isolate was identified from a co-culture of marine bacteria-bacteria.
Macrolides Derived from the Co-Cultures of Different Marine Bacteria A known compound, nonactin (100) (Figure 17) was isolated from the co-culture of two marine bacteria, Saccharomonospora sp. UR22 and Dietzia sp. UR66 [51]. It possessed a macrotetrolide structure integrated from nonactic acid, and exhibited antitumor and antibacterial activity, especially

Macrolide
There were no reported macrolides from the co-cultures of marine fungi-fungi and fungi-bacteria. Only one isolate was identified from a co-culture of marine bacteria-bacteria.

Macrolides Derived from the Co-Cultures of Different Marine Bacteria
A known compound, nonactin (100) (Figure 17) was isolated from the co-culture of two marine bacteria, Saccharomonospora sp. UR22 and Dietzia sp. UR66 [51]. It possessed a macrotetrolide structure integrated from nonactic acid, and exhibited antitumor and antibacterial activity, especially its inhibitory effects on the P170 glycoprotein-mediated efflux of chemotherapeutic agents in multiple-drug-resistant cancer cells [62][63][64][65].

Macrolide
There were no reported macrolides from the co-cultures of marine fungi-fungi and fungibacteria. Only one isolate was identified from a co-culture of marine bacteria-bacteria.

Macrolides Derived from the Co-Cultures of Different Marine Bacteria
A known compound, nonactin (100) (Figure 17) was isolated from the co-culture of two marine bacteria, Saccharomonospora sp. UR22 and Dietzia sp. UR66 [51]. It possessed a macrotetrolide structure integrated from nonactic acid, and exhibited antitumor and antibacterial activity, especially its inhibitory effects on the P170 glycoprotein-mediated efflux of chemotherapeutic agents in multiple-drug-resistant cancer cells [62][63][64][65].

Phenylpropanoids
Phenylpropanoids are a big and structurally diverse group of secondary metabolites, which bear a C6-C3 phenolic scaffold that play crucial roles in a wide spectrum of biological and pharmacological

Phenylpropanoids
Phenylpropanoids are a big and structurally diverse group of secondary metabolites, which bear a C 6 -C 3 phenolic scaffold that play crucial roles in a wide spectrum of biological and pharmacological activities [66]. Twenty-three phenylpropanoids were isolated from co-culture of marine fungi-fungi (12 isolates, 52%) and fungi-bacteria (11 isolates, 48%), while there are no reported phenylpropanoids from the co-culture of different marine bacteria.
Ten citrinin analogues were isolated and identified from the co-culture of two marine algalderived endophytic fungal strains, Aspergillus sydowii EN-534 and Penicillium citrinum EN-535 collected from marine red alga Laurencia okamurai, including two novel compounds, citrinin dimer seco-penicitrinol A (102) and citrinin monomer penicitrinol L(103), and the known penicitrinone A (104), penicitrinone F (105), penicitrinol A (106), citrinin (107), dihydrocitrinone (108), decarboxydihydrocitrinone (109) phenol A acid (110) and phenol A (111) (Figure 19) [68]. In addition, one novel coumarin named 7-(γ,γ-dimethylallyloxy)-6-hydroxy-4-methylcoumarin (112) (Figure 19) was detected and characterized from the co-culture of the two mangrove fungi, Phomopsis sp. K38 and Alternaria sp. E33 [69].  . Sterigmatocystin (120) also exhibited strong cytotoxicity towards human hepatoma cells (HepG2) at 3 µM [72]. Its mechanism suggested that it could stimulate a biotransformation process, increase the population of reactive oxygen species and promote the imbalance in the antioxidant defense system caused by the process of lipid peroxidation [73]. Recently, Zingales et al. (2020) displayed the significant role of mitochondria in sterigmatocystin-induced toxicity in SH-SY5Y cells [74]. The reduced viability of SH-SY5Y cells displayed time-and dose-dependence with mitochondrial dysfunction when exposed to 120 in response to the forced dependency of the cells on mitochondrial oxidative phosphorylation [74]. Thus, these findings provided us a valuable direction for the application of neuroprotective mitochondria-target functional peptides. Moreover, compound 122 inhibited human umbilical vein endothelial cells (VEGF-induced proliferation of HUVECs) with an IC 50 value of 1.4 µM [75]. It is considered as a novel inhibitor of vascular endothelial cell growth factor, which is one of the main stimulants of angiogenesis. and E. ictaluri with MIC values of 32-64 μg/mL. 103 and 105 inhibited V. parahaemolyticus and E. coli with MIC values of 32 and 64 μg/mL, respectively. Moreover, 102-107 were further evaluated for anti-influenza neuraminidase (homologous protein of H5N1) activity. 104 and 105 exhibited significant inhibitory activities with IC50 values of 12.9 and 18.5 nM, respectively [68]. Thus, these bioactive substances could be further optimized for the development of antibacterial and antiinfluenza agents. In addition to the anti-influenza activity, the activated metabolite penicitrinone A (104) also exerted an inhibitory effect on four human cancer cell lines, HL-60, K562, BGC-823 and HeLa cells with IC50 values of 43.2, 50.8, 54.2 and 65.6 μM, respectively [70].

Phenylpropanoids Derived from the Co-Cultures of Marine Fungi and Bacteria
The chemical investigation of the mixed culture of the marine fungus A. versicolor and B. subtilis resulted in the isolation of one novel aflaquinolone, 22-epi-aflaquinolone B (113); and ten known metabolites, aflaquinolone A, F and G (114-116) (Figure 20) [38].  . Sterigmatocystin (120) also exhibited strong cytotoxicity towards human hepatoma cells (HepG2) at 3 μM [72]. Its mechanism suggested that it could stimulate a biotransformation process, increase the population of reactive oxygen species and promote the imbalance in the antioxidant defense system caused by the process of lipid peroxidation [73]. Recently, Zingales et al. (2020) displayed the significant role of mitochondria in sterigmatocystin-induced toxicity in SH-SY5Y cells [74]. The reduced viability of SH-SY5Y cells displayed time-and dose-dependence with mitochondrial dysfunction when exposed to 120 in response to the forced dependency of the cells on mitochondrial oxidative phosphorylation [74]. Thus, these findings provided us a valuable direction for the application of neuroprotective mitochondria-target functional peptides. Moreover, compound 122 inhibited human umbilical vein endothelial cells (VEGF-induced proliferation of HUVECs) with an IC50 value of 1.4 μM [75]. It is considered as a novel inhibitor of vascular endothelial cell growth factor, which is one of the main stimulants of angiogenesis.

Polyketides
Twelve polyketides were isolated and characterized from the marine microbial co-cultures in recent years (Figures 2B and 21).

Polyketides
Twelve polyketides were isolated and characterized from the marine microbial co-cultures in recent years ( Figures 2B and 21).  [76]. Two unprecedented polyketides (127-128) had a common feature-a conjugated carboxylic acid group that could be biogenetically generated from the methyl group of an acetate rather than a methionine precursor in 127, and the same group could be derived from C-1 position of an acetate or C-2 position of a propionate in 128 based on the precursor of the ethyl group connected to a double bond. It was an excellent case of a truly novel carbon skeleton induced by the powerful and underexplored method, marine microbial  (Figure 22) from the marine-derived fungi Penicillium sp. Ma(M3)V isolated from the marine sponge Mycale angulosa co-cultivated with Trichoderma sp. Gc(M2)1 isolated from the marine sponge Geodia corticostylifera [76]. Two unprecedented polyketides (127-128) had a common feature-a conjugated carboxylic acid group that could be biogenetically generated from the methyl group of an acetate rather than a methionine precursor in 127, and the same group could be derived from C-1 position of an acetate or C-2 position of a propionate in 128 based on the precursor of the ethyl group connected to a double bond. It was an excellent case of a truly novel carbon skeleton induced by the powerful and underexplored method, marine microbial co-cultivation. sterigmatocystin (124), 5-methoxysterigmatocystin (125) and aversin (126) (Figure 22) from the ethyl acetate extract of two marine alga-derived fungi, Aspergillus sp. BM-05 and BM-05ML [30]. Kossuga et al. isolated two new and unusual polyketides: (Z)-2-ethylhex-2-enedioic acid (127) and (E)-4-oxo-2-propylideneoct-7-enoic acid (128) (Figure 22) from the marine-derived fungi Penicillium sp. Ma(M3)V isolated from the marine sponge Mycale angulosa co-cultivated with Trichoderma sp. Gc(M2)1 isolated from the marine sponge Geodia corticostylifera [76]. Two unprecedented polyketides (127-128) had a common feature-a conjugated carboxylic acid group that could be biogenetically generated from the methyl group of an acetate rather than a methionine precursor in 127, and the same group could be derived from C-1 position of an acetate or C-2 position of a propionate in 128 based on the precursor of the ethyl group connected to a double bond. It was an excellent case of a truly novel carbon skeleton induced by the powerful and underexplored method, marine microbial co-cultivation.  Both 129 and 130 exhibited moderate inhibitory activity against H1975 tumor cell lines with IC50 values of 3.97 and 5.73 μM, respectively. Deoxyfunicone (131) was found to exert anti-inflammatory activity, exhibiting the inhibition effect on overproduction of nitric oxide (NO) and the prostaglandin E2 in both lipopolysaccharide-provoked BV2 microglial and lipopolysaccharide-stimulated RAW264.7 macrophage cells (IC50 = 10.6 and 40.1 μM, respectively) [78]. 132 was known as a cytotoxic, genotoxic, mutagenic and fetotoxic mycotoxin [79,80]. However, in the IL-1β-stimulated Caco-2 cells, the metabolite 132 increased the transcription of TNF-α; inversely reduced the transcription of IL-1β and IL-6; and decreased the transcription and secretion of IL-8, suggesting that 132 possessed immunomodulatory activities on both lipopolysaccharide-and IL-1 β-related pathways in non-immune intestinal epithelial cells [79].

Polyketides Derived from the Co-Cultures of Different Marine Bacteria
Recently, two unusual polyketides, janthinopolyenemycins A (134) and B (135) (Figure 24) were purified and identified from the co-cultivation broth of two marine bacteria Janthino bacterium spp. ZZ145 and ZZ148 isolated from marine soil sample [81]. Both 134 and 135 displayed the same antifungal activity against C. albicans with a minimum bactericidal concentration (MBC) value of 31.25 μg/mL and an MIC value of 15.6 μg/mL. However, none of them could suppress the growth of methicillin-resistant S. aureus or E. coli (MIC > 100 μg/mL) [81]. Both 129 and 130 exhibited moderate inhibitory activity against H1975 tumor cell lines with IC 50 values of 3.97 and 5.73 µM, respectively. Deoxyfunicone (131) was found to exert anti-inflammatory activity, exhibiting the inhibition effect on overproduction of nitric oxide (NO) and the prostaglandin E 2 in both lipopolysaccharide-provoked BV2 microglial and lipopolysaccharide-stimulated RAW264.7 macrophage cells (IC 50 = 10.6 and 40.1 µM, respectively) [78]. 132 was known as a cytotoxic, genotoxic, mutagenic and fetotoxic mycotoxin [79,80]. However, in the IL-1β-stimulated Caco-2 cells, the metabolite 132 increased the transcription of TNF-α; inversely reduced the transcription of IL-1β and IL-6; and decreased the transcription and secretion of IL-8, suggesting that 132 possessed immunomodulatory activities on both lipopolysaccharide-and IL-1 β-related pathways in non-immune intestinal epithelial cells [79].

Polyketides Derived from the Co-Cultures of Different Marine Bacteria
Recently, two unusual polyketides, janthinopolyenemycins A (134) and B (135) (Figure 24) were purified and identified from the co-cultivation broth of two marine bacteria Janthino bacterium spp. ZZ145 and ZZ148 isolated from marine soil sample [81]. Both 134 and 135 displayed the same antifungal activity against C. albicans with a minimum bactericidal concentration (MBC) value of 31.25 µg/mL and an MIC value of 15.6 µg/mL. However, none of them could suppress the growth of methicillin-resistant S. aureus or E. coli (MIC > 100 µg/mL) [81]. RAW264.7 macrophage cells (IC50 = 10.6 and 40.1 μM, respectively) [78]. 132 was known as a cytotoxic, genotoxic, mutagenic and fetotoxic mycotoxin [79,80]. However, in the IL-1β-stimulated Caco-2 cells, the metabolite 132 increased the transcription of TNF-α; inversely reduced the transcription of IL-1β and IL-6; and decreased the transcription and secretion of IL-8, suggesting that 132 possessed immunomodulatory activities on both lipopolysaccharide-and IL-1 β-related pathways in non-immune intestinal epithelial cells [79].

Polyketides Derived from the Co-Cultures of Different Marine Bacteria
Recently, two unusual polyketides, janthinopolyenemycins A (134) and B (135) (Figure 24) were purified and identified from the co-cultivation broth of two marine bacteria Janthino bacterium spp. ZZ145 and ZZ148 isolated from marine soil sample [81]. Both 134 and 135 displayed the same antifungal activity against C. albicans with a minimum bactericidal concentration (MBC) value of 31.25 μg/mL and an MIC value of 15.6 μg/mL. However, none of them could suppress the growth of methicillin-resistant S. aureus or E. coli (MIC > 100 μg/mL) [81].

Steroids
Steroids contain a characteristic arrangement of four cycloalkane rings that are joined together. They represent a large family of compounds that play important roles as chemical messengers, and the scaffold is present in many FDA-approved drugs [82][83][84]. A total of five steroidal metabolites were reported; four of them were isolated from the co-culture of marine fungi-bacteria (80%); only one isolate was identified from the co-culture of marine fungi-fungi (20%). No isolates were obtained from the co-culture of marine bacteria-bacteria.

Steroids
Steroids contain a characteristic arrangement of four cycloalkane rings that are joined together. They represent a large family of compounds that play important roles as chemical messengers, and the scaffold is present in many FDA-approved drugs [82][83][84]. A total of five steroidal metabolites were reported; four of them were isolated from the co-culture of marine fungi-bacteria (80%); only one isolate was identified from the co-culture of marine fungi-fungi (20%). No isolates were obtained from the co-culture of marine bacteria-bacteria.

Steroids Derived from the Co-Cultures of Different Marine Fungi
To the best of our knowledge, the only one steroid, ergosterol (136), was found from the co-culture broth of two marine mangrove epiphytic fungi, Aspergillus sp. FSY-01 and FSW-02 ( Figure 25) [21,85]. It was an essential component of fungal cell membrane with strong specificity and stable structure. Therefore, 136 was widely applied to detecting fungal containment as an indicator of fungal biomass [86]. To the best of our knowledge, the only one steroid, ergosterol (136), was found from the coculture broth of two marine mangrove epiphytic fungi, Aspergillus sp. FSY-01 and FSW-02 ( Figure 25) [21,85]. It was an essential component of fungal cell membrane with strong specificity and stable structure. Therefore, 136 was widely applied to detecting fungal containment as an indicator of fungal biomass [86].

Steroids Derived from the Co-Cultures of Marine Fungi and Bacteria
An unprecedented steroid, 7β-hydroxycholesterol-1β-carboxylic acid (137), together with three known steroidal metabolites, 7β-hydroxycholesterol (138), 7α-hydroxycholesterol (139) and ergosterol-5α,8α-peroxide (140) (Figure 26), have been confirmed from the co-culture of two marine alga-derived microbes, Aspergillus sp. BM05, and an unidentified bacterium (BM05BL), isolated from the brown alga of the genus Sargassum collected off Helgoland, North Sea, Germany [87]. Compounds 137-140 showed moderate activities against four human tumor cell lines, A2780, HCT116, K562 and A2780 CisR with the IC50 values of 10.0-100.0 μM. At the same time, the total extract of co-culture of Aspergillus sp. BM05 and BM05BL showed obvious antiproliferative activity compared with its single steroidal compounds. This implied a synergistic role of these steroidal metabolites in the extract. Furthermore, 140 was reported as a promising new candidate that could overcome the drug-resistant property of malignant cancer cells through abolishing miR-378, a microRNA involved in new tumor initiation, unlimited self-renewal and recurrence of tumor cells after chemotherapy [88].

Terpenoids
Terpenoids known as isoprenoids are structurally diverse metabolites found in many natural sources. This class of compounds displays a wide sector of important pharmacological entities that confirmed by several preclinical and clinical studies [89,90]. Only two terpenoidals were isolated from the co-cultures of marine fungi-bacteria (one compound, 50%) and bacteria-bacteria (one compound, 50%).

Terpenoids Derived from the Co-Cultures of Marine Fungi and Bacteria
The production of the bacterial sesquiterpene pentalenic acid (141) (Figure 27) might be attributed to the competition relationship between marine fungus A. fumigatus MR2012 isolated from a Red Sea sediment in Hurghada, Egypt and terrestrial bacterium S. leeuwenhoekii C58 collected from Compounds 137-140 showed moderate activities against four human tumor cell lines, A2780, HCT116, K562 and A2780 CisR with the IC 50 values of 10.0-100.0 µM. At the same time, the total extract of co-culture of Aspergillus sp. BM05 and BM05BL showed obvious antiproliferative activity compared with its single steroidal compounds. This implied a synergistic role of these steroidal metabolites in the extract. Furthermore, 140 was reported as a promising new candidate that could overcome the drug-resistant property of malignant cancer cells through abolishing miR-378, a microRNA involved in new tumor initiation, unlimited self-renewal and recurrence of tumor cells after chemotherapy [88].

Terpenoids
Terpenoids known as isoprenoids are structurally diverse metabolites found in many natural sources. This class of compounds displays a wide sector of important pharmacological entities that confirmed by several preclinical and clinical studies [89,90]. Only two terpenoidals were isolated from the co-cultures of marine fungi-bacteria (one compound, 50%) and bacteria-bacteria (one compound, 50%).

Terpenoids Derived from the Co-Cultures of Marine Fungi and Bacteria
The production of the bacterial sesquiterpene pentalenic acid (141) (Figure 27) might be attributed to the competition relationship between marine fungus A. fumigatus MR2012 isolated from a Red Sea sediment in Hurghada, Egypt and terrestrial bacterium S. leeuwenhoekii C58 collected from the hyper-arid soil of Laguna de Chaxa Salar de Atacama, Chile, in which S. leeuwenhoekii C58 suppressed the production of A. fumigatus MR2012 and enhanced the production of 141 [37]. This suggested that S. leeuwenhoekii C58 appeared to activate the cryptic biosynthetic gene clusters to construct a defense mechanism based on the chemical signals generated by the competitive fungus, A. fumigatus MR2012. Thus, the bacterial strain was capable of suppressing the biosynthesis of the fungus metabolites that were present in the axenic cultures.
Mar. Drugs 2020, 18, x 20 of 27 the hyper-arid soil of Laguna de Chaxa Salar de Atacama, Chile, in which S. leeuwenhoekii C58 suppressed the production of A. fumigatus MR2012 and enhanced the production of 141 [37]. This suggested that S. leeuwenhoekii C58 appeared to activate the cryptic biosynthetic gene clusters to construct a defense mechanism based on the chemical signals generated by the competitive fungus, A. fumigatus MR2012. Thus, the bacterial strain was capable of suppressing the biosynthesis of the fungus metabolites that were present in the axenic cultures.

Terpenoids Derived from the Co-Cultures of Different Marine Bacteria
A diterpene lobocompactol (142) (Figure 28) was isolated from the co-culture of marine actinomycete Streptomyces cinnabarinus PK209 collected from the seaweed rhizosphere, obtained at a depth of 10 m along the coast of Korea and its competitor Alteromonas sp. KNS-16. Its productivity was increased 10.4-fold higher than that of the pure culture of PK209 [91]. Moreover, its antifouling activities were recently confirmed against primary fouling organisms, including diatoms, bacteria, and macroalgae zoospores. In order to further determine whether 142 was a non-toxic antifoulant, the therapeutic rate (LC50/EC50) was used to evaluate its toxicity, the LC50/EC50 of 142 was more than that of 15, indicating that the metabolite 142 was a non-toxic antifoulant. Thus, this compound could be valuable as an antifouling agent in both antifouling coating industry and marine ecology.

Terpenoids Derived from the Co-Cultures of Different Marine Bacteria
A diterpene lobocompactol (142) (Figure 28) was isolated from the co-culture of marine actinomycete Streptomyces cinnabarinus PK209 collected from the seaweed rhizosphere, obtained at a depth of 10 m along the coast of Korea and its competitor Alteromonas sp. KNS-16. Its productivity was increased 10.4-fold higher than that of the pure culture of PK209 [91]. Moreover, its antifouling activities were recently confirmed against primary fouling organisms, including diatoms, bacteria, and macroalgae zoospores. In order to further determine whether 142 was a non-toxic antifoulant, the therapeutic rate (LC 50 /EC 50 ) was used to evaluate its toxicity, the LC 50 /EC 50 of 142 was more than that of 15, indicating that the metabolite 142 was a non-toxic antifoulant. Thus, this compound could be valuable as an antifouling agent in both antifouling coating industry and marine ecology. was increased 10.4-fold higher than that of the pure culture of PK209 [91]. Moreover, its antifouling activities were recently confirmed against primary fouling organisms, including diatoms, bacteria, and macroalgae zoospores. In order to further determine whether 142 was a non-toxic antifoulant, the therapeutic rate (LC50/EC50) was used to evaluate its toxicity, the LC50/EC50 of 142 was more than that of 15, indicating that the metabolite 142 was a non-toxic antifoulant. Thus, this compound could be valuable as an antifouling agent in both antifouling coating industry and marine ecology.
Among the five diorcinols, only 146 showed apparent cytotoxicity against murine Ehrlich carcinoma cells and hemolytic activity against mouse erythrocytes. The significant hemolytic activity of 146 suggested that its cytotoxic activity against murine Ehrlich carcinoma cells was due to a membranolytic mechanism. It is well known that the heat shock protein 70 (HSP70) was frequently overexpressed in tumor cell lines as an ATP-dependent molecular chaperone and played a significant role in refolding misfolded proteins and promoting cell survival under stress [94]. Thus, compounds that could inhibit HSP70 had great potential in tumor therapy. 147 could decrease the expression of HSP70 in the Ehrlich carcinoma cells, which made it possible to develop as a new antitumor drug/lead. Diorcinol D (149) was studied for its combined therapy against planktonic Candida albicans with a broad-spectrum antifungal agent fluconazole [95]. The combined therapy exhibited considerable antifungal activity against ten clinical isolates of C. albicans containing five fluconazoleresistant isolates and five fluconazole-sensitive isolates, whereas fluconazole alone did not display antifungal activity. This suggested that diorcinol D (149) restored the susceptibility of fluconazole to C. albicans.
Moreover, the efficiencies of fluconazole inhibiting mature biofilms were also drastically boosted by the addition of 149 [95]. The fractional inhibitory concentration index (FICI) model and Compound 143 showed in vitro inhibitory activity against G. musae, F. graminearum, P. sojae (Kaufmann and Gerdemann) and Rhizoctonia solani Kuhn at 0.25 mM with inhibition zone diameters of 11.57, 12.06, 8.5 and 10.21 mm, respectively. This suggested that 143 had broad inhibitory activity against these microbes [92]. 144 and 145 exhibited weak toxicity against brine shrimp (LC 50 > 100 µM) and none of them displayed cytotoxicity against the liver hepatocellular carcinoma Huh7 and HepG2 (LC 50 > 100 µM) and obvious inhibitory activities towards three marine-derived bacteria, Bacillus stearothermophilus, Pseudoalteromonas nigrifaciens and Bacillus amyloliquefaciens, and two common pathogens, P. aeruginosa and S. aureus [23].
Among the five diorcinols, only 146 showed apparent cytotoxicity against murine Ehrlich carcinoma cells and hemolytic activity against mouse erythrocytes. The significant hemolytic activity of 146 suggested that its cytotoxic activity against murine Ehrlich carcinoma cells was due to a membranolytic mechanism. It is well known that the heat shock protein 70 (HSP70) was frequently overexpressed in tumor cell lines as an ATP-dependent molecular chaperone and played a significant role in refolding misfolded proteins and promoting cell survival under stress [94]. Thus, compounds that could inhibit HSP70 had great potential in tumor therapy. 147 could decrease the expression of HSP70 in the Ehrlich carcinoma cells, which made it possible to develop as a new antitumor drug/lead. Diorcinol D (149) was studied for its combined therapy against planktonic Candida albicans with a broad-spectrum antifungal agent fluconazole [95]. The combined therapy exhibited considerable antifungal activity against ten clinical isolates of C. albicans containing five fluconazole-resistant isolates and five fluconazole-sensitive isolates, whereas fluconazole alone did not display antifungal activity. This suggested that diorcinol D (149) restored the susceptibility of fluconazole to C. albicans.
Moreover, the efficiencies of fluconazole inhibiting mature biofilms were also drastically boosted by the addition of 149 [95]. The fractional inhibitory concentration index (FICI) model and ∆E model unclosed that the synergistic actions indeed existed in combination of diorcinol D (149) and fluconazole [95]. Two resistance mechanisms of azoles were overexpression of efflux pumps genes and alterations of genes (point mutations). 149 mainly suppressed the activity of efflux pump in cells partly by decreasing the expression of Cdr1 (one mediator of azole efflux pumps) in Candida albicans CASA1. On the other hand, 149 also inhibited ergosterol synthesis and CYP51 (the target of fluconazole) expression [95]. Thus, the significant synergistic interaction and drug-resistant reversion of fluconazole combined with diorcinol D (149) were caused by the two latent mechanisms, the block of efflux pump and ergosterol biosynthesis. Notably, 149 was still needed to further in vivo study in the combination therapy field to settle rock-ribbed clinical fungal infection in response to the azole resistance. Compounds 149, 152 and 153 displayed antibacterial activities against five Gram-positive microbes, including one S. aureus, two E. faecalis and two E. faecium with the MIC values of 12.5-50 μM. In addition, 152 displayed potent inhibitory activities against all tested bacteria with an MIC value of 12.5 μM. 149 displayed inhibitory activity against E. coli with an MIC value of 8 μg/mL; and 153 showed significant antibacterial activity against S. aureus with an MIC value of 6.25 μg/mL [96,97]. In contrast, 149, 152 and 153 did not display any obvious activity against L5178Y cell lines, which suggested that the antimicrobial activities of these products were not associated with their respective general toxicities [38].

Conclusions
Marine microorganisms have attracted more attention as natural producers of lead compounds. Marine microbes especially are considered as a renewable and reproducible source that can be easily cultured [98,99]. However, the speed of new lead compound discovery is slowing down. Thus, marine microbial co-culturing represents a powerful strategy for the production of novel biosubstances. The strategy can induce the biosynthesis of novel compounds and various NPs coded by corresponding genomes through the activation of the silent gene clusters or previously unexpressed biosynthetic routes.
In the last ten years, the overall statistical studies showed that 156 metabolites were discovered from the co-culture of different marine microbes. Figure 2 and Table 1 illustrated that 59 compounds were isolated from the co-culturing of different marine fungi; 79 compounds were isolated from marine fungi and bacteria; and only 18 compounds were disclosed from co-culturing different marine bacteria. The metabolites by co-culture of marine fungi and bacteria accounted for the largest proportion (51% of all metabolites of marine microbial co-culture). Alkaloids were the largest group with ≥51.9%, whereas macrolides were the lowest group with <0.65%. Just only one macrolide was identified from the co-cultures of different marine bacteria. Furthermore, co-cultures of different marine bacteria did not produce cyclopeptides, phenylpropanoids and steroids, and co-cultures of different marine fungi did not induce the biosynthesis of terpenoids.
Several studies suggest that Aspergillus spp. are the most common fungi that co-fermented with other microbes and produce numerous novel skeletons. The majority of these NPs have antimicrobial Compounds 149, 152 and 153 displayed antibacterial activities against five Gram-positive microbes, including one S. aureus, two E. faecalis and two E. faecium with the MIC values of 12.5-50 µM. In addition, 152 displayed potent inhibitory activities against all tested bacteria with an MIC value of 12.5 µM. 149 displayed inhibitory activity against E. coli with an MIC value of 8 µg/mL; and 153 showed significant antibacterial activity against S. aureus with an MIC value of 6.25 µg/mL [96,97]. In contrast, 149, 152 and 153 did not display any obvious activity against L5178Y cell lines, which suggested that the antimicrobial activities of these products were not associated with their respective general toxicities [38].

Conclusions
Marine microorganisms have attracted more attention as natural producers of lead compounds. Marine microbes especially are considered as a renewable and reproducible source that can be easily cultured [98,99]. However, the speed of new lead compound discovery is slowing down. Thus, marine microbial co-culturing represents a powerful strategy for the production of novel bio-substances. The strategy can induce the biosynthesis of novel compounds and various NPs coded by corresponding genomes through the activation of the silent gene clusters or previously unexpressed biosynthetic routes.
In the last ten years, the overall statistical studies showed that 156 metabolites were discovered from the co-culture of different marine microbes. Figure 2 and Table 1 illustrated that 59 compounds were isolated from the co-culturing of different marine fungi; 79 compounds were isolated from marine fungi and bacteria; and only 18 compounds were disclosed from co-culturing different marine bacteria. The metabolites by co-culture of marine fungi and bacteria accounted for the largest proportion (51% of all metabolites of marine microbial co-culture). Alkaloids were the largest group with ≥51.9%, whereas macrolides were the lowest group with <0.65%. Just only one macrolide was identified from the co-cultures of different marine bacteria. Furthermore, co-cultures of different marine bacteria did not produce cyclopeptides, phenylpropanoids and steroids, and co-cultures of different marine fungi did not induce the biosynthesis of terpenoids.
Several studies suggest that Aspergillus spp. are the most common fungi that co-fermented with other microbes and produce numerous novel skeletons. The majority of these NPs have antimicrobial or/and antitumor activities. However, some significant restrictions obstruct the development of the co-culture technology; e.g., cryptic and undefined biosynthesis routes and the producers of NPs from the co-cultivation of two or more microorganisms, the particularities of strains and environmental and nutritional requirements, the instability of the ecological relationship, the uncertainty of the interaction relationship and the high contamination probability. Therefore, new technology and equipment need to be created, such as metabolomics analysis and molecular network technology. The new mechanisms of chemical communication of microbes (through direct/mediate contact) also need to be further investigated. In conclusion, co-culture is still shrouded in mystery as a prospective experimental tool for novel bioactive NPs. This article embodies the value and diversity of NPs from the co-cultivation of marine-derived microorganisms and it is considered as a guided reference for studying NPs.

Conflicts of Interest:
The authors declare no conflict of interest.